Genome-wide investigation of the PLD gene family in alfalfa (Medicago sativa L.): identification, analysis and expression

Background External environmental factors, such as salt, alkali and drought, severely limit the acreage and yield of alfalfa. The mining of tolerance-related genes in alfalfa and improving the stress resistance of this plant are essential for increasing alfalfa yield. PLD is the main phospholipid hydrolase in plants and plays an important role in plant growth, development, signaling, and resistance to adverse stress. With the availability of whole genome sequences, the annotation and expression of PLDs in alfalfa can now be achieved. At present, few studies have investigated PLDs in alfalfa. Here, we conducted a study of PLDs in alfalfa and identified and analyzed the expression pattern of PLDs under different treatments. Results Fifty-nine MsPLDs were identified in alfalfa and classified into six subtypes: MsPLDα, β, γ, δ and ε belong to the C2-PLD subfamily, and MsPLDζ belongs to the PXPH-PLD subfamily. Members of the same PLD subtype have similar physicochemical properties, sequence structure and domains, but their cis-acting elements are different. A qRT-PCR analysis revealed that MsPLDs are expressed in multiple tissues. MsPLDs can respond to alkali, drought, ABA, IAA, and GA3 treatments and particularly to salt stress. Different expression patterns were found for the same gene under different treatments and different genes under the same treatment. Expression of MsPLD05 improved salt tolerance in yeast. Conclusion This study represents the first genome-wide characterization of MsPLDs in alfalfa. Most MsPLDs are expressed mainly in mature leaves and respond positively to abiotic stresses and hormonal treatments. This study further expands the resistance gene pool in legume forage grasses and provides a reference for further in-depth study of MsPLDs in alfalfa. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-022-08424-9.


Introduction
Phospholipids are not only an important component of cell membranes but also an important source of intracellular signal generation [1]. Phospholipids can be hydrolyzed by phospholipases to produce phosphatidic acid (PA), choline, ethanol and diacylglycerol (DAG). Phospholipases are classified as phospholipase Al (PLA1), phospholipase A2 (PLA2), phospholipase B (PLB), phospholipase C (PLC) and phospholipase D (PLD) depending on the hydrolysis site of glycerophospholipids [1,2]. Different types of phospholipases show differences in terms of reaction conditions, cofactors and substrate selection [1,2]. Phospholipase D is the most important phospholipase in plants, can specifically catalyze the hydrolysis of phosphodiester bonds at the end of phospholipid molecules and participates in processes such as plant growth and development [3,4].
Currently, PLDs have been identified in rice (Oryza sativa) [5], Arabidopsis (Arabidopsis thaliana) [6], soybean (Glycine max) [7], grape (Vitis vinifera), poplar (Populus L.) [8] and Chinese plum (Prunus mume) [9] plants, which have more than 10 PLDs. In Arabidopsis, the PLD gene family is divided into six isoforms based on information on their physicochemical properties and sequence structures: PLDα, β, γ, δ, ε and ζ. However, the N-terminal structural domains of PLDs show differences: PLDα, β, γ, δ, and ε contain a C2 structural domain at the N-terminal end and comprise the C2-PLD subfamily, whereas PLDζ has a PX/ PH structural domain at the N-terminal end and forms the PX/PH-PLD subfamily. The C2 structural domain is involved in Ca 2+ -dependent and phospholipid binding and all members except PLDα2, β2, γ3 and PLDζ require Ca 2+ activation, but the amount of Ca 2+ needed varies among genes [6,10,11]. In addition, the PX structural domain is essential for the activity of PLDs in the PLDζ subfamily [12,13].
Several studies have shown that PLDs are involved in the production of PAs that are important in plant growth, development, signaling, resistance to adverse stress. Studies on the function of AtPLDs have revealed that the functions exercised by different PLD isoforms may vary [14,15]. AtPLDα and δ members are involved in responses to abiotic stresses such as salt and drought [16][17][18][19][20][21][22]. In addition, AtPLDα1 and AtPLDδ are involved in stomatal closure, cell senescence, and cell death [21]. AtPLDε promotes primary root and root hair elongation under low nitrogen conditions [23,24]. AtPLDβ is associated with the defense response to fungal pathogen infestation [25]. This finding further suggests that different PLDs perform unique and important functions in specific plant growth, development or stress response processes. A summary of the mechanism of action of PLDs reveals that PLDs mainly function by altering the membrane lipid composition, degrading the membrane mass, disrupting membrane function, and participating in cellular regulation as signaling molecules [26]. The most in-depth studies on PLDα1 in Arabidopsis mainly focused on PLDprotein interactions, including direct protein interactions and protein interactions through PA and other products. At present, some PLD target proteins have been identified, and these include the heterotrimeric G protein Gα subunit (Gα) [27] and the aspartate protease cardosin A [28]. The identified PA target proteins include NADPH oxidase [29], phosphatidylinositol-dependent protein kinase 1 (PDK1) [30], mitogen-activated protein kinase 6 (MPK6) [31], and sphingosine kinase (SPHK) [32]. The abovementioned studies reveal that the PLD-and PArelated signaling pathways form an important component of the plant phospholipid signaling pathway.
Alfalfa is a perennial legume herb with good palatability, high nutritional value and high yield and is known as the "queen of forage grasses" [33]. This herb is also used as an ecological grass to prevent soil erosion and improve soil quality due to its well-developed root system and nitrogen fixation ability [34]. Therefore, alfalfa is widely planted in many countries. Although the PLD gene family has been identified in many plants, no comprehensive study of PLDs in alfalfa has been reported thus far. With the release of the alfalfa genome [35], we are better able to systematically investigate the putative functions of PLDs in alfalfa. As a result, we successfully identified 59 MsPLDs in alfalfa and analyzed the basic physicochemical properties, evolutionary tree, sequence structure, structural domains, and covariance of these 59 MsPLDs. These MsPLDs were also analyzed in terms of cis-acting elements and by qRT-PCR to further clarify their possible functions. The results will lay the foundation for further study of these 59 MsPLDs and the mining of resistance gene resources.

Identification and characterization of MsPLDs in alfalfa
To identify and obtain the MsPLDs in the alfalfa genome, a global search of the alfalfa genome using the hidden Markov model (HMM) profile of the HKD domain (PF000164) sequence alignment, conserved structural domain analysis and other methods identified a total of 59 MsPLDs, and these MsPLDs were used for subsequent analyses (Table 1). Their protein sequences and coding sequences are listed in Additional file 1. MsPLD01-59 were renamed according to their position on the chromosome, and the names and IDs of the genes are presented in Table 1.
The main information regarding the gene locations, subcellular localizations and physicochemical properties of the proteins are also presented in Table 1. The MsPLDs were unevenly distributed on all chromosomes except chromosome 6. The analysis of the distribution of the genes revealed that chromosomes 1, 7 and 8 had a sparser distribution, with 2, 4 and 4 MsPLDs, respectively, whereas each of the remaining chromosomes had more than 10 MsPLDs. Subcellular localization predictions showed that most of the MsPLDs were distributed in the cytoplasm, followed by the vacuole, endoplasmic reticulum and chloroplast. Therefore, we hypothesized that MsPLDs mainly exercise functions in the cytoplasm. The analysis of the physicochemical properties of the proteins relative differences in terms of their sequence length, isoelectric point (pI) and molecular weight within the ranges of 607 to 1125 aa, 5.5 to 8.31 and 68.53 to 128.59 kDa, respectively. More than half of the sequences had a length of 800-900 aa. With the

Phylogenetic and protein sequence analysis of the MsPLD gene family
To investigate the evolutionary relationships of MsPLD gene family members, a phylogenetic tree was constructed using amino acid sequences of Arabidopsis and alfalfa ( Fig. 1). The results showed that the 59 MsPLDs in alfalfa were divided into six isoforms, namely MsPLDα, β, γ, δ, ε, and ζ. This result was consistent with findings in Arabidopsis, where the maximum number of MsPLDα was 16, and the minimum number of MsPLDζ and MsPLDε were 6 in alfalfa. The diversity of the gene structures supported phylogenetic grouping to some extent [36]. To better show the sequence structure of MsPLDs in alfalfa, we constructed a phylogenetic evolutionary tree using the protein sequences of 59 MsPLDs (Fig. 2a). The comparison of the exon-intron organizations of different MsPLDs showed that the gene structures of MsPLDs were relatively different but similar among the same isoforms (Fig. 2b). Among the MsPLDs, gene members belonging to the MsPLDβ, γ and δ subtypes all contain 8 or 9 introns. In contrast, the number of introns in the MsPLDα members ranged from 1 to 4. With the exception of MsPLD56, all other members of the MsPLDε subtype contained 3 introns. The maximum number of introns obtained for the MsPLDζ subtype was between 18 and 20. We speculate that this finding is related to the longer sequences of the MsPLDζ subtype members.
To further investigate the structural features of alfalfa PLD proteins, the conserved motifs were analyzed. A total of 10 conserved motifs were obtained (Fig. 2c). The logos and basic information of these base sequences are shown in Additional files 2 and 3. All members of the MsPLDα, β, γ, δ and ε subtypes contained motifs 1-10 with the exception of MsPLD03, which did not contain motifs 2 and 7, and MsPLD48, which did not harbor motif 8. In contrast, the members of the MsPLDζ subtype contained only motifs 1, 4, 5, 6 and 9. A CDD analysis revealed that the 59 MsPLDs contained a total of 7 domains, and all of these proteins were related to phospholipase D (Fig. 2d). The subsequent analysis using the Pfam and InterPro websites revealed that the PLDζ subtype members all contained the PXPH structural domain and classified these members into the PXPH-PLD subfamily, whereas the remaining five subtypes all harbored the C2 structural domain and were classified into the C2-PLD subfamily. Subsequent sequence alignment revealed that the two HKD structural domains were highly conserved (except for the mutation of D in the second HKD structural domain of MsPLD03 to K and the deletion of D in the second HKD structural domain of MsPLD56) and separated by 271-400 amino acids (Additional file 4). The structures predicted for the two PLD subfamilies are shown in Fig. 2e. These results are consistent with the classification results obtained in species such as Arabidopsis. We thus hypothesize that PLDs are highly conserved in plants and the location of each structural domain varies from species to species.

Collinearity and gene duplication analysis of MsPLDs
Gene duplication events have played an important role in the expansion of many gene families; therefore, a homologous BLAST of amino acids of alfalfa was performed with the MCScan toolkit, and 139,504 synteny gene pairs and 9733 groups of tandem duplication genes were identified at the genome level in alfalfa. The MsPLD gene family of synteny gene pairs and tandem duplication gene pairs were selected and visualized using TBtools ( Fig. 3 and Additional file 5). In the MsPLD gene family of alfalfa, 59 synteny gene pairs including 4 pairs of segmental duplication gene events, were identified. In addition, 8 tandem duplication events occurred. Information on these gene pairs is provided in Additional file 5. Replication events regarding PLDs have not been reported in Arabidopsis. Thus, the findings demonstrate that gene duplication events enabled expansion of the MsPLD gene family in alfalfa, which may also explain why the number of PLDs in alfalfa is markedly higher than that in Arabidopsis. The Ka, Ks and Ka/Ks of the MsPLD gene pairs were calculated to study the evolutionary functional constraints in alfalfa (Additional file 6). In the present study, the Ka values for each gene pair were found to be in the range of 0 to 0.083 whereas Ks values ranged from 0 to 0.80. All of the MsCSase gene pairs with Ka/Ks > 1 were subjected to positive selection. The results suggest that purifying selection was the main force driving the evolution of MsPLDs.

Regulatory elements in the MsPLD promoters
To identify putative cis-elements involved in MsPLD transcriptional regulation, a 2.0-kb promoter region upstream from the ATG translation start codon of each MsPLD was analyzed. We selected 24 major putative cis-elements and grouped them into three categories: 6 Fig. 1 Phylogenetic analysis of PLDs in alfalfa and Arabidopsis. The amino acid sequences of PLD proteins from alfalfa and Arabidopsis were used for the phylogenetic analysis. The phylogenetic tree was constructed with MEGA 6.0 using the maximum likelihood method with 1000 bootstrap replicates. The different colors on the evolutionary tree branches represent different PLD subfamilies, and the symbols ○, △ and ◇ represent PLDs in alfalfa, Arabidopsis, and Drosophila, respectively cis-elements that respond to stress, 8 cis-elements that respond to hormones, and 10 light-responsive elements (Fig. 4). DRE, MYB and MYC are typical promoter ciselements involved in abiotic stress induction in plants and can positively respond to stresses such as high salt, low temperature and drought. The hormone-responsive elements identified were associated with hormones such as salicylic acid, anxin, gibberelline and abscisic acid. In addition, MYB, MYC, ARE, ABRE, G-box, GT1 motif and Box 4 were the most abundant. However, no clear correlation was found between the type and number of cis-acting elements and the distribution of subfamily members. Thus, we speculate that MsPLDs may be involved in abiotic stress and hormonal regulation, and different members of the same subfamily may have different response patterns.

qRT-PCR analysis of MsPLDs in different samples
To further clarify the potential functions that may exist for MsPLDs, we performed a qRT-PCR analysis of different samples of alfalfa. First, twelve MsPLDs were selected from different subfamilies based on the phylogenetic analysis and the analysis of the cis-acting elements in the promoter region. The experimental results (Fig. 5) showed that the 12 selected MsPLDs were expressed in the roots, stems and leaves of alfalfa. These results are consistent with findings in Arabidopsis [23,37]. PLDs can be expressed in multiple plant tissues. MsPLD47 and MsPLD59 were mainly expressed in roots, whereas the other 10 MsPLDs were mainly expressed in leaves, and the levels of the same gene showed differences between young and mature leaves. MsPLD05 was similarly expressed at higher levels in stems and young leaves. This finding may be related to the fact that different genes act in different tissues.
Based on the results from the cis-acting element analysis and studies of the PLD gene family in Arabidopsis, we determined the relative expression of 12 MsPLD genes under various abiotic stresses and different hormone treatments. A qRT-PCR analysis under salt, alkali, and drought stresses revealed that MsPLDs responded to abiotic stresses through different expression patterns (Fig. 6a-c). Most of the MsPLDs responded to drought and salt stresses via upregulation (Fig. 6a, c), whereas under alkali stress, most MsPLDs showed a downregulated expression pattern (Fig. 6b). Under salt stress, all MsPLDs except MsPLD07 and MsPLD13 showed the highest expression at 3 h, whereas no obvious change was detected under drought and alkali stresses. A comparison of the expression patterns among different subfamily members under the same stress treatment revealed that most subfamily members presented similar expression patterns, for example, MsPLDα, β, δ and ζ under drought stress, MsPLDα, γ, δ and ζ under alkali stress and MsPLDβ, γ, δ and ζ under salt stress. The remaining subfamily members exhibited similar expression patterns among the differential expression patterns of the remaining subfamily members and need to be further investigated. The expression pattern of MsPLD05 was most representative of the three stresses: the relative expression of this gene significantly upregulated under salt stress, was not significantly altered under drought stress but was significantly downregulated in response to alkali stress.
To better understand the role of MsPLDs in phytohormone signaling pathways, a qRT-PCR analysis of alfalfa under ABA, IAA and GA3 hormone treatments ( Fig. 7a-c) was performed, and the results revealed that the MsPLD07, MsPLD13, MsPLD51, MsPLD55, MsPLD59, and MsPLD41 genes could respond positively to these three hormone treatments. With the exception of the downregulated trend observed for MsPLD11 under GA3 treatment, all the responding genes roughly showed upregulated expression, but the expression levels of same genes under different hormone treatments and those of different genes under the same hormone

Expression of MsPLD05 improves salt tolerance in yeast
Combining the results of sequence analysis and qRT-PCR experiments, we finally cloned MsPLD05 and expressed it in yeast. Under normal growth conditions, there was no significant phenotypic difference between yeast containing pESC-HIS and pESC-HIS-MsPLD05. However, under salt stress, pESC-HIS-MsPLD05 yeast had a better phenotype, and the phenotypic differences became more pronounced with increasing salt concentrations (Fig. 8). This result also implies that this gene can respond positively to this stress and can improve stress resistance when expressed.

Discussion
Phospholipase D is the most important class of phospholipid hydrolases in plants and plays an important role in regulating cell membrane lipid metabolism, participating in plant growth and development and responding to stress [3,4,18,20,29,38]. To date, most studies on PLDs have focused on responses to specific stresses, such as salt, cold, and ABA. However, the identification of PLDs in alfalfa and their response under multiple stresses have not been reported. Therefore, in this study, the PLDs in alfalfa were identified and analyzed with respect to their bioinformatics and expression patterns under various stresses. The present study has furthered our understanding of MsPLDs and provides insight into the functions of MsPLDs.

Evolution of the alfalfa MsPLD gene family
The number of PLDs varies among species. Fewer than 20 PLDs have been identified in other species, such as Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa L.), and apple (Malus × domestica) [5,9,39]. A total of 59 MsPLDs were identified in the alfalfa genome. The number of PLDs in alfalfa was markedly higher than that in other diploid plants. We speculate that this result was obtained because alfalfa is a tetraploid plant, whereas all ither abovementioned plants are diploid; in addition, our identification results may include alleles, which would, in turn, leads to an excess number of PLDs in alfalfa. In addition, a comparison of the gene duplication events of PLDs (Fig. 3) revealed more tandem duplication events and synteny duplication events in alfalfa, which also directly leads to an increase in the number of PLDs in alfalfa.
The 59 PLDs in alfalfa were classified into six isoforms, MsPLDα, β, γ, δ, ε, and ζ, and into two subfamilies according to differences in their structural domains (Fig. 1). Members of the same isoform have similar sequence lengths, physicochemical properties, gene structures and motif distributions; however, differences in these features were observed among different isoforms, and the largest differences were detected between members of the MsPLDζ isoform and the other isoforms (Table 1, Fig. 2b-d). This finding may be related to differences in the N-terminal structural domain. These results also further support the results from the evolutionary analysis. Although the gene classification results were consistent with the results obtained in Arabidopsis, the gene structural domain analysis revealed that the two were more different, with 271-407 amino acids between the two HKD structural domains in alfalfa (Fig. 2e) and approximately 320 amino acids between the two HKD structural domains in Arabidopsis [6,39]. This finding suggests that although PLDs are highly conserved in plants, they show differences among species, but whether these differences lead to new functions of PLDs in alfalfa needs to be further investigated. Duplication events in the active regions, the coding sequence (CDS), and/or the regulatory sequence, the promoter region, can cause members of a gene family to acquire new functions [40,41].

Potential roles of MsPLDs in alfalfa
The tissue-specific expression analysis of MsPLDs revealed that the 12 selected genes were expressed in different tissues, but the majority of the MsPLDs were highly expressed in leaves, and only MsPLD47 and MsPLD59 were mainly expressed in roots (Fig. 5). This finding also implies that MsPLD47 and MsPLD59 may exercise their functions mainly in the roots. Interestingly, the subcellular localization prediction shows that MsPLD59 is localized in chloroplasts and the cytoplasm (Table 1). It has been shown that the subcellular localization of genes is affected by the growth period of plants, and different developmental stages are specifically expressed in different tissues [42]. Whether this finding is also the case for MsPLD59 and whether MsPLD59 functions in alfalfa roots need to be further verified. In addition, the relative expression of the same gene showed differences between young and mature leaves, which suggests that MsPLDs regulate the growth and development of alfalfa leaves through different expression patterns at different developmental periods. Overall, most of the MsPLDs were localized in the leaves, where they exert their actions.
We performed a qRT-PCR analysis of alfalfa under abiotic stress and hormone treatments, and this expression pattern analysis can provide insight into the potential functions of the MsPLD gene family. The experimental results showed that PLDs could respond positively to abiotic stress and hormone treatments, and the expression patterns of PLDs exhibited differences between different treatments (Figs. 6 and 7). The MsPLDs responded most significantly to salt stress among the different treatments, which was similar to the findings obtained in Arabidopsis. The exposure of Arabidopsis to salt stress activates and induces PLDα1 to produce PA in the plant, and this step is followed by activation of related enzymes downstream of PA [20,31,[43][44][45], whereas AtPLDα3 regulates the salt stress response by promoting root growth [37]. This finding shows that different PLDs have different modes of action in response to salt stress. Based on this result, we hypothesized that different MsPLDs may have different modes of action under the same stress, which subsequently leads to different expression patterns. Thus, whether the same genes that show different expression patterns when subjected to different stresses also have different modes of action needs to be further investigated.
In the present study, we found that MsPLDs show differences in terms of gene structure, predicted subcellular localization, and expression patterns. Moreover, PLDs can be activated in different ways, have distinguishable functions in Arabidopsis, and are involved in plant growth, development, and responses to multiple stresses. A model summarizing MsPLDs in response to abiotic stress and hormonal treatments is shown in Fig. 9, and this model is based on the results from previous studies [15,46] as well as the present study. Different PLDs in alfalfa plants exhibit different expression patterns after abiotic stress and hormone treatments. PLDs can directly bind to effector proteins such as GAPC, Gα, and actin and indirectly interact with PA effector proteins such as protein kinases, protein phosphatase, and lipid kinase through PA, which can jointly participate in relevant physiological processes after stress and improve the plant's ability to adapt to variable environments.

Conclusion
In conclusion, a total of 59 MsPLDs were identified in the alfalfa genome, and these 59 MsPLDs were divided into 6 subtypes based on their phylogenetic relationships and 2 subfamilies based on their structural domains. Members of the same isoform have similar physicochemical properties, sequence structure, and domains. Duplications have likely been important for the expansion and evolution of MsPLDs. These MsPLDs contain cis-acting elements that respond to abiotic stress, hormones and light. A qRT-PCR analysis showed that most MsPLDs could respond positively to abiotic stress and hormone treatments, particularly to salt stress. To our knowledge, this study involves the first systematic and in-depth analysis of alfalfa MsPLDs, and these data provide a foundation for elucidating the molecular mechanism of MsPLDs underlying stress biology. This study expands the genetic resources for improving plant tolerance and serves as a reference for future functional investigations and molecular breeding in alfalfa.

Identification and phylogenetic analysis of alfalfa MsPLDs
First, we obtained the hidden Markov model of the PF00164 domain from the Pfam 34.0 database (https:// pfam. xfam. org/). The genome assembly files of alfalfa (cultivar XinJiangDaYe) [35] were downloaded from the website (https:// figsh are. com/ proje cts/ whole_ genome_ seque ncing_ and_ assem bly_ of_ Medic ago_ sativa/ 66380). We searched the alfalfa genome using HMMER 3.0 software [47] and used online sites such as Pfam [48] and InterPro (http:// www. ebi. ac. uk/ inter pro/) for screening and identification. The protein sequences of the 12 PLDs of Arabidopsis were obtained from The Arabidopsis Information Resource (https:// www. arabi dopsis. org/ index. jsp). A phylogenetic tree was generated with

Nucleotide and base sequence analysis
The chromosome location and sequence information of MsPLDs in alfalfa was obtained from the website (https:// figsh are. com/ proje cts/ whole_ genome_ seque ncing_ and_ assem bly_ of_ Medic ago_ sativa/ 66380). Protein sequences were analyzed using the ExPASy database (https:// prosi te. expasy. org/) to obtain information such as the relative molecular mass, amino acid length and theoretical isoelectric point. The protein sequences were analyzed for motifs and domains using MEME Suite 5.3.3 (https:// meme-suite. org/ meme/ index. html) [49] and NCBI (https:// www. ncbi. nlm. nih. gov/), respectively. All the analytical results were visualized using TBtools software [50]. In addition, amino acid sequence alignment was performed with DNAMAN. The cis-acting element of the promoter region 2000 bp upstream of the CDS region of MsPLDs was predicted using the PlantCARE (http:// bioin forma tics. psb. ugent. be) online analysis website [51].

Collinearity and multiple synteny analysis of MsPLDs
The MCScanX tool was used to identify all tandem blocks and colinear gene pairs in the alfalfa genome, and relevant information about MsPLDs was extracted and visualized using TBtools [50]. In addition, the synonymous substitution rate (Ks) and nonsynonymous substitution rate (Ka) of MsPLDs and the Ks/Ka ratio were calculated with TBtools [50].

Plant materials and treatments
In this study, alfalfa (cultivar XinJiangDaYe) was used as the plant material. Alfalfa seeds with uniform morphology and full seeds were selected, cultured in vermiculite and watered with 1/10 Hoagland nutrient solution. All plants were grown under a 16-h light/8-h dark photoperiod with day/night temperatures of 22 °C/18 °C. When the seeds were 4 weeks of age, plants with uniform growth were treated with 15% PEG-6000, 150 mmol/L NaCl, 150 mmol/L NaHCO 3 , 100 mmol/L ABA, 100 mmol/L IAA and 100 mmol/L GA3. Samples were collected at 0 h (CK) and 1 h, 3 h, 6 h, 12 h, and 24 h after treatment. Samples of roots, stems, mature leaves and young leaves were collected from one-month-old alfalfa for tissuespecific expression analysis. All sample collections were set up with three biological replicates. The samples were snap-frozen in liquid nitrogen after sampling, transferred to -80 °C and stored for subsequent analysis.

Validation of MsPLD expression levels by qRT-PCR
Total RNA extraction and reverse transcription of the collected plant samples were performed based on the instructions provided with the kit. The quality of the reverse-transcribed cDNA was examined using the GAPDH gene as an internal reference. The reaction system was 1 µl of ddH 2 O, 5 µl of 2 × Phanta Max Master Mix, 1 µl of GAPDH S (10 µM), 1 µl of GAPDH AS (10 µM) and 1 µl of cDNA. The samples were loaded on the PCR system and subjected to the following conditions: 180 s at 95 °C followed by 40 amplification cycles consisting of denaturation for 10 s at 95 °C, annealing for 30 s at 58 °C, and extension for 30 s at 72 °C. The qRT-PCR experiments were performed using high-quality cDNA and 2 × Cham Q Universal SYBR q PCR Master Mix, and the rest of the reaction system and conditions were consistent with those used in the PCR analysis. Each reaction was performed with three replicates. The sequences of the internal reference primers are shown in Additional file 7. The relative levels of gene expression were determined by the 2 -ΔΔCT method [52].

Yeast expression vector construction and the salt tolerance of MsPLD05
In this experiment, the coding sequence of the MsPLD05 gene was obtained by PCR using the cDNA that was reverse transcribed from RNA extracted from alfalfa leaves under normal growth conditions. The MsPLD05 fragment was ligated to pESC-HIS using T4 DNA ligase, and the recombinant expression plasmid was obtained and named pESC-HIS-MsPLD05. pESC-HIS and pESC-HIS-MsPLD05 were transformed into brewer's yeast Y187 using the LiAc/SS-DNA/PEG transformation method [53]. The recombinant strains were screened on nutrient-deficient SD-HIS medium. Yeast strains transformed with pESC-HIS and pESC-HIS-MsPLD05 were isolated and then used for gradient dilution and inoculation into SD-HIS medium with NaCl (0, 0.5 and 1 mol/L). The results were obtained after 2 days of culture at 29 °C. The sequences of the primers are shown in Additional file 7.

Statistical analysis
Statistical analysis was performed using SPSS statistics 22.0 software. Data were subjected to analysis of variance, and the means were compared using Student's t-test at the 5% significance level.